Vaccine formulations

ABSTRACT

A vaccine containing one or more synthetic or highly purified natural peptides or proteins as antigen(s) as well as one or more adjuvants is present in the form of a solution or emulsion which is free from inorganic salt ions or has a low concentration of salt ions. Preferably, it contains substances capable of making the vaccine isotonic, particularly sorbitol.

The present invention relates to the field of vaccines.

The immunogenic effect of traditional vaccines is mostly based onpathogens which have been killed or attenuated. In traditional vaccinesthe impurities in the vaccines themselves or other components oforganisms act as adjuvants which potentiate and/or prolong theimmunogenic activity of the actual antigen. For example, thediphtheria-tetanus-whooping cough vaccine contains two potent adjuvantsoriginating from the whole-cell whooping cough vaccine(LPS=lipopolysaccharide and PT=pertussis toxin); Similarly, the wholecell typhus and cholera vaccines have potent adjuvants (LPS and choleratoxin); the BCG vaccine (Bacillus Calmette Guerin) has powerfulnon-specific immunostimulatory effects.

In contrast to the complex traditional vaccines, modern vaccines containsynthetic, recombinant or highly purified antigens in the form ofproteins or peptides. These vaccines are regarded as safer but generallyhave the disadvantage of lower immunogenicity. To compensate for thisdisadvantage, adjuvants are added to the vaccines, to increase andprolong the specific immune response to antigens. Some adjuvants havethe property of intensifying T-cell proliferation and the cellularimmune response.

Most of the adjuvants used hitherto have side effects, however, andfurthermore these adjuvants do not meet the requirements imposed on thesafety of adjuvants, such as stability with respect to adjuvantactivity, minimal toxicity with no interaction with the antigen, andalso degradability in the body and the absence of any immunogenicactivity of their own.

A summary of current adjuvants which have hitherto been considered foruse in vaccines is provided by Vogel, 1995, and by Gupta and Siber,1995. They include: inorganic adjuvants in gel form (aluminiumhydroxide/aluminium phosphate, calcium phosphate); bacterial adjuvantssuch as monophosphoryl lipid A and muramyl peptides, particulateadjuvants such as the so-called ISCOMS (“immunostimulatory complexes”),liposomes and biodegradable microspheres, adjuvants based on oilemulsions and emulsifiers such as Freund's adjuvant or IFA (“IncompleteFreund's adjuvant”), saponines (such as QS-21), squalene; syntheticadjuvants such as non-ionic block copolymers, muramyl peptide analogues,synthetic lipid A, synthetic polynucleotides and polycationic adjuvantssuch as polyarginine or polylysine (WO 97/30721).

The choice of an adjuvant is usually a compromise which is the result ofbalancing the toxicity and adjuvant effect of the substance in question.

In vaccine formulations, care has generally been taken up to now toachieve isotonicity; the common vaccine formulations are usually in asalt concentration which corresponds to about 150 mM of NaCl (about 300mosmol/l). Common buffer formulations are PBS and HBS(phosphate-buffered or HEPES-buffered saline); e.g. for an ISCOM vaccinePBS pH 7.4 was proposed (Barr and Mitchell, 1996).

The aim of the present invention was to provide a vaccine formulationwhich intensifies the activity of vaccines based on antigens in the formof peptides or proteins.

It was found that, surprisingly, the immunogenic activity of apeptide-based vaccine containing adjuvant is increased if the vaccineformulation has a low concentration of salt ions or is free from salts.

The invention thus relates to a vaccine containing one or more syntheticor highly purified natural peptides or proteins as antigen(s) as well asone or more adjuvants. The vaccine is characterised in that it takes theform of a solution or emulsion which is free from inorganic salt ions orhas a low concentration of salt ions.

In the context of the vaccine according to the invention the phrase “lowconcentration of salt ions” denotes a concentration which is equal to orless than about 50% of the salt concentration of an isotonic solution,corresponding to about 75 mM saline solution.

For calculating the ion concentration it should be borne in mind that,when using peptide or protein antigens which themselves have a charge,this charge is not taken into account.

Preferably, the vaccine is substantially free from sodium, chloride andphosphate ions, and particularly preferably it is substantially freefrom all inorganic salt ions (“substantially free” means that no saltshave been added to the vaccine, but that there may be impurities presentwhich have originated from reagents or there may be traces of ions; ionsoriginating from adjuvants are not included in the calculation either,e.g. when using inorganic adjuvants).

In the event that the vaccine contains phosphate ions, e.g. originatingfrom buffer solution, it is preferably free from sodium and chlorideions. If it contains sodium and/or chloride ions, it is preferably freefrom phosphate ions.

In one embodiment of the invention the vaccine contains antigen andadjuvant in salt-free medium, e.g. in distilled water.

In another preferred embodiment the vaccine according to the inventioncontains one or more water-soluble or water-emulsifiable substanceswhich are capable of making the vaccine isotonic and increasing itsimmunogenic activity.

These substances are hereinafter designated “isotonic-makingsubstances”. Isotonic-making substances have the property of being ableto generate physiological osmotic pressure by virtue of their molecularsize and molecular structure.

Preferably, the isotonic-making substances are selected from among thegroup carbohydrates (sugars, sugar alcohols, oligosaccharides,polysaccharides), polyhydric alcohols, amino acids or lipids.

Preferably, the isotonic-making substance is a sugar, particularly amono- or disaccharide such as maltose, fructose, galactose orsaccharose, or a sugar alcohol such as sorbitol or mannitol.

The amino acids used may be isotonic, salt-free amino acid solutionssuch as are used e.g. in parenteral feeding. Solutions of this kind arecommercially obtainable (e.g. from Leopold, Graz, Austria); if necessarythey may be desalinated if they contain salt ions. Alternatively,isotonic, salt-free solutions which contain individual, preferablywater-soluble, amino acids may be used.

The lipids used may be, in particular, isotonic, salt-free fattyemulsions such as those used in parenteral feeding, for example.Emulsions of this kind are commercially obtainable (e.g. from Leopold,Graz, Austria); if necessary they may be desalinated if they containsalt ions. It is also possible to use long-chain hydrocarbons (e.g.paraffin oils), and also higher fatty acids such as linoleic acid,linolenic acid or palmitic acid, and fatty acid esters such astriglycerides.

The isotonic-making substance is preferably present in a concentrationsuch that the resulting solution is isotonic or slightly hypotonic,depending on the molecular weight.

Preferred sugar or sugar alcohol concentrations are within the rangefrom about 200-400 mM, particularly in the range from 250-300 mM. Theosmolarity of the solution is conveniently between 200-400 mosmol/l, butthe solution may also be strongly hypotonic.

Amino acid solutions should preferably have an osmolarity of between200-400 mosmol/l, but may also be strongly hypotonic.

Lipid emulsions also preferably have an osmolarity of between 200-400mosmol/l, but may also be strongly hypotonic.

In addition to the isotonic-making substance the solution comprising thevaccine according to the invention optionally contains a buffersubstance. This might be, in particular, HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]), or TRIS(tris[hydroxymethyl]aminomethane). A buffer substance may be necessaryto adjust the vaccine to a physiological pH if the primary solution isdifferent from the physiological value.

The vaccine according to the invention is not subject to anyrestrictions regarding the peptide or protein antigens. The antigens maybe naturally occurring immunogenic proteins, e.g. proteins from viral orbacterial pathogens or the fragments thereof or cellular breakdownproducts in the form of peptides; or tumour antigens or fragmentsthereof. In a preferred embodiment the antigen is a tumour antigen or anatural or synthetic peptide derived therefrom; in this case the vaccineis a tumour vaccine.

The quantity of effective antigen in the vaccine according to theinvention may vary over a wide range.

The quantity of peptide depends, among other things, on the method ofadministration and the particular formulation used. The amount ofpeptide to be administered may be about 0.1 μg to about 10000 μg pervaccination dose, generally 1.0 μg to about 1000 μg, particularly about10 μg to about 500 μg.

In a preferred embodiment of the invention the adjuvant is a substancesuch as that proposed in WO 97/30721, the disclosure of which isexpressly referred to here, as an additive for protein or peptidevaccines, preferably a polycation such as polyarginine or polylysinewhich is optionally modified, e.g. with a sugar group.

The adjuvant used may also be, theoretically, any of the abovementionedadjuvants known for peptide- or protein-based vaccines. For example:inorganic adjuvants in gel form (aluminium hydroxide/aluminiumphosphate, Warren et al., 1986; calcium phosphate, Relyvelt, 1986);bacterial adjuvants such as monophosphoryl lipid A (Ribi, 1984; Baker etal., 1988) and muramyl peptides (Ellouz et al., 1974; Allison and Byars,1991; Waters et al., 1986); particulate adjuvants such as the so-calledISCOMS (“immunostimulatory complexes”, Mowat and Donachie, 1991;Takahashi et al., 1990; Thapar et al., 1991), liposomes (Mbawuike et al.1990; Abraham, 1992; Phillips and Emili, 1992; Gregoriadis, 1990) andbiodegradable microspheres (Marx et al., 1993); adjuvants based on oilemulsions and emulsifiers such as Freund's adjuvant or IFA (“IncompleteFreund's adjuvant” (Stuart-Harris, 1969; Warren et al., 1986), SAF(Allison and Byars, 1991), saponines (such as QS-21; Newman et al.,1992), squalene/squalane (Allison and Byars, 1991); synthetic adjuvantssuch as non-ionic block copolymers (Hunter et al., 1991), muramylpeptide analogues (Azuma, 1992), synthetic lipid A (Warren et al., 1986;Azuma, 1992), synthetic polynucleotides (Harrington et al., 1978) andpolycationic adjuvants (WO 97/30721).

The skilled person will be able to define suitable antigen/adjuvantformulations from the specialist literature mentioned hereinbefore and,working from this starting point, find an isotonic-making substancewhich is capable of increasing the efficacy of the formulation or, whileretaining the same efficacy, reducing the proportion of adjuvant in theformulation, which offers a critical advantage in the case of adjuvantswith side effects.

It has surprisingly been found, within the scope of the presentinvention, that a salt-free tumour vaccine made isotonic with sorbitol,containing an MHC-binding peptide derived from a tumour antigen as wellas polyarginine as adjuvant, has a more potent antitumour activity thana conventionally formulated tumour vaccine, i.e. containing an isotonicsalt concentration, which is identical in terms of the peptide/adjuvant.It was found that the peptides together with the adjuvant dissolvebetter in sorbitol solution than in conventional PBS buffer. Withoutwishing to be tied to the theory, the improved activity of the vaccine,apart from the improved solubility, would appear to be due to the factthat the interaction between the peptide and adjuvant is made easier andthus the activity of the adjuvant is intensified. The improved activityof the vaccine may possibly also be due to a co-adjuvant activity of theisotonic-making substance, e.g. sorbitol, i.e. this substance (sorbitol)as such has a certain adjuvant effect which increases the activity ofthe primary adjuvant.

The following method is appropriately used to achieve the ideal vaccineformulation: starting from a defined antigen, which is intended toprovoke the desired immune response, in a first step an adjuvant matchedto the antigen is found, as described in the specialist literature,particularly in WO 97/30721. In a next step the vaccine is optimised byadding various isotonic-making substances as defined in the presentinventions, preferably sugars and/or sugar alcohols, in an isotonic orslightly hypotonic concentration, to the mixture of antigen andadjuvant, with the composition otherwise being identical, and adjustingthe solution to a physiological pH in the range from pH 4.0 to 10.0,particularly 7.4. Then, in a first step as described in the example ofthe present application, the substances or the concentration thereofwhich will improve the solubility of the antigen/adjuvant compositioncompared with a conventional, saline-buffered solution are determined.The improvement in the solubility characteristics by a candidatesubstance is a first indication that this substance is capable ofbringing about an increase in the immunogenic activity of the vaccine.

Since one of the possible prerequisites for an increase in the cellularimmune response is increased binding of the antigen to APCs (antigenpresenting cells), in a next step an investigation can be made to seewhether the substance leads to an increase of this kind. The procedureused may be analogous to that described in the definition of theadjuvant, e.g. incubating APCs with fluorescence-labelled peptide orprotein, adjuvant and isotonic-making substance. An increased uptake orbinding of the peptide to APCs brought about by the substance can bedetermined by comparison with cells which have been mixed with peptideand adjuvant alone or with a peptide/adjuvant composition which ispresent in conventional saline buffer solution, using throughflowcytometry.

In a second step the candidate substances may be investigated in vitroto see whether and to what extent their presence is able to increase thepresentation of a peptide to APCs; the MHC concentration on the cellsmay be measured using the methods described in WO 97/30721 for testingpeptides.

Another possible way of testing the efficiency of a formulation is byusing an in vitro model system. In this, APCs are incubated togetherwith adjuvant, peptide and candidate substance and the relativeactivation of a T-cell clone which specifically recognises the peptideused is measured (Coligan et al., 1991; Lopez et al., 1993).

The efficiency of the formulation may optionally also be demonstrated bythe cellular immune response by detecting a “delayed-typehypersensitivity” (DTH) reaction in immunised animals.

Finally, the immunomodulatory activity of the formulation is measured inanimal tests. In the case of a tumour vaccine as in the present example,established tumour models having known peptide sequences recognised byimmune cells may be used, inter alia. The vaccine, containing differentbuffer substances but having a constant peptide/adjuvant composition, isadministered to the test animals. The protection from tumour growth is ameasurement of the efficacy of a tumour vaccine.

EXAMPLE

The experiments were carried out as described in WO 97/30721.

a) DBA/2 mice were inoculated three times at intervals of one week witha mixture of 100 μg of MHC Class I binding peptide SYFPETHI (SEQ IDNO: 1) (known as “P815 JAK1”) and 75 μg of polyarginine (degree ofpolymerisation 70, SIGMA Chemicals, St. Louis Mo.) per animal. Thepeptide/adjuvant solution was administered in sorbitol solution (270 mMsorbitol, 5 mM HEPES) or phosphate-buffered saline solution (PBS, GIBCOBRL). Control mice were either given 100 μg of peptide/animal with noadjuvant in sorbitol buffer or were not vaccinated. A week after thelast vaccination, 104 viable tumour cells were injected and tumourgrowth was measured weekly.

The results of the tests are shown in FIG. 1. The Figure shows acomparison of the efficiency of the P815 JAK1 vaccine in sorbitolsolution as against a vaccine in buffered isotonic saline solution inthe animal model. It was found that animals that had been given thevaccine in sorbitol solution were better protected than mice that hadbeen inoculated with peptide/polyarginine in BS.

b) For the solubility tests, mixtures of fluorescence-labelled peptideLFEAIEGFI (SEQ ID NO:2) or GYKDGNEYI (SEQ ID NO:2) were prepared: 100 μgof fluorescence-labelled peptide were combined with 75 μg ofpolyarginine (Arg; degree of polymerisation 70, SIGMA Chemicals, St.Louis Mo.) either in sorbitol solution or HEPES-buffered saline solution(HBS: 20 mM HEPES pH 7.5, 150 mM NaCl). After three hours the amount ofdissolved fluorescence was measured by determining the extinction at 490nM. The test protein used was Green Fluorescent protein.

FIG. 2 and FIG. 3 show a comparison of the solubility of the complexesafter mixing in buffered saline solution or sorbitol solution. The twofluorescence-labelled peptides (FIG. 2A and FIG. 2B) and the GreenFluorescent protein (GFP; about 30 Kd; FIG. 3) were included in thisexperiment. Adding the vaccine in sorbitol solution resulted in asignificantly better solubility and recovery (increased fluorescence)both with the two tested peptides and with GFP.

LITERATURE

-   Abraham, E., 1992, Vaccine 10, 461-468-   Allison, A. C., and Byars, N. E., 1991, Mol Immunol 28, 279-284-   Azuma, I., 1992, Vaccine 10, 1000-1004-   Baker, P. J., et al., 1988, Infect Immun 56, 3064-3066-   Coligan, J. E. et al., 1991, Current Protocols in Immunology, Wiley,    New York-   Ellouz, F., et al., 1974, Biochem Biophys Res Commun 59, 1317-1325-   Gupta, R. K. and Siber G. R., 1995, Vaccine 13, 1263-1276-   Gregoriadis, G., 1990, Immunol Today 11, 89-97-   Harrington, D. G., et al., 1978, Infect Immun 24, 160-166-   Hunter, R., et al., 1991, Vaccine 9, 250-255-   Lopez, J. A., et al., 1993, Eur. J. Immunol. 23, 217-223-   Marx, P. A., et al., 1993, Science 28, 1323-1327-   Mbawuike, I. N., et al., 1990, Vaccine 8, 347-352-   Mowat, A. M., and Donachie, A. M., 1991, Immunol Today 12, 383-385-   Newman, M. J., et al., 1992, J Immunol 148, 23572362-   Phillips, N. C. and Emili, A, 1992, Vaccine 10, 151-158-   Rammensee, H. G., et al., 1995, Immunogenetics 41, 178-228-   Relyvelt, E. H., 1986, Develop Biol Standard, 65, 131-136-   Ribi, E., 1984, J Biol Res Mod, 3, 1-9-   Stuart-Harris, C. H., 1969, Bull WHO 41, 617-621-   Takahashi, H., et al., 1990, Nature 344, 873-875-   Thapar, M. A., et al., 1991, Vaccine 9, 129-133-   Vogel, F. R. 1995, Ann N Y Acad Sci 754, 153-160-   Warren, H. S., et al., 1986, Ann Rev Immunol 4, 369-388-   Waters, R. V., et al., 1986, Infect Immun 52, 816-825

1. Vaccine containing one or more synthetic or purified natural peptidesor proteins as antigen(s) as well as one or more adjuvants,characterised in that it is present as a solution or emulsion which issubstantially free from inorganic salt ions, wherein said solution oremulsion contains one or more water soluble or water-emulsifiablesubstances which is capable of making the vaccine isotonic or hypotonic,said substance(s) selected from the group consisting of: a) a maltose;b) a fructose; c) a galactose; d) a saccharose; e) a sugar alcohol; f) alipid; and g) combinations thereof, wherein at least one of saidadjuvants is a polycation, optionally modified with a sugar group. 2.Vaccine according to claim 1, characterized in that said water solubleor water-emulsifiable substance is present in a concentration such thatthe resulting solution is isotonic.
 3. Vaccine according to claim 1,characterized in that it additionally contains a buffer.
 4. Vaccineaccording to claim 1, characterized in that it contains a peptide as theantigen.
 5. Vaccine according to claim 4, characterized in that thepeptide is a tumour antigen or a fragment thereof and is capable ofbinding to MHC molecules.
 6. Vaccine according to claim 1, characterizedin that it contains polyarginine as the adjuvant.
 7. Vaccine accordingto claim 1, characterized in that said water soluble orwater-emulsifiable substance is present in a concentration such that theresulting solution is hypotonic.
 8. The vaccine formulation of claim 1,wherein said water soluble or water-emulsifiable substance is a sugaralcohol.
 9. The vaccine formulation of claim 1, wherein said watersoluble or water-emulsifiable substance is maltose.
 10. The vaccineformulation of claim 1, wherein said water soluble or water-emulsifiablesubstance is fructose.
 11. The vaccine formulation of claim 1, whereinsaid water soluble or water-emulsifiable substance is galactose.
 12. Thevaccine formulation of claim 1, wherein said water soluble orwater-emulsifiable substance is saccharose.
 13. Vaccine according toclaim 8, characterized in that the sugar alcohol is mannitol.
 14. Thevaccine formulation of claim 1, wherein said water soluble orwater-emulsifiable substance is a lipid.
 15. The vaccine formulation ofclaim 1, wherein said water soluble or water-emulsifiable substances areselected from the group consisting of maltose, fructose, galactose,saccharose and combinations thereof.
 16. The vaccine formulation ofclaim 15, characterized in that the concentration of the water solubleor water-emulsifiable substances is in the range from about 200-400 mM.17. The vaccine formulation of claim 16, characterized in that theconcentration of the water soluble or water-emulsifiable substances isin the range from about 250-300 mM.
 18. The vaccine formulation of claim1, characterized in that the concentration of the water soluble orwater-emulsifiable substances is in the range from about 200-400 mM. 19.The vaccine formulation of claim 18, characterized in that theconcentration of the water soluble or water-emulsifiable substances isin the range from about 250-300 mM.
 20. The vaccine according to claim8, wherein said sugar alcohol is sorbitol.
 21. The vaccine according toclaim 8, characterized in that the concentration of sugar alcohol is inthe range from about 200-400 mM.
 22. The vaccine according to claim 20,characterized in that the concentration of sorbitol is 250-300 mM. 23.The vaccine according to claim 1, wherein said vaccine is in solutionform.
 24. The vaccine according to claim 1, wherein said vaccine is inemulsion form.
 25. The vaccine according to claim 23, wherein saidadjuvant is selected from the group consisting of polyarginine andpolylysine.
 26. The vaccine according to claim 25, wherein said adjuvantis polylysine.
 27. The vaccine according to claim 26, wherein saidisotonic making substance sugar alcohol.
 28. The vaccine according toclaim 27, wherein said sugar alcohol is sorbitol.